Most of the endothelium in the body is continuous endothelium (endothelial layer), and continuous endothelium is observed in the blood-brain barrier, diaphragm, muscular tissue of the duodenum, fat, heart, papillary microvascular system, large vessel, lung, mesentery, nerve, blood-retina barrier, skeletal muscle, testis, and in or in the vicinity of another tissue or organ in the body. The continuous endothelium forms a semi-permeable membrane forming a barrier between tissue or organs contacting each other and permitting permeation of water, ions, a small molecule, a macromolecule, and cells by a regulated scheme. Various diseases may be caused by abnormal permeation of materials in the endothelial layer. Alternatively, as the disease progresses, abnormal permeation of materials in the endothelial layer may be generated. As an example, it was known that abnormal permeation in the brain microvascular endothelial cell layer is closely associated with initiation and progression of brain diseases such as Alzheimer's disease, stroke, and multiple sclerosis.
Further, in the case of injecting a drug into a specific tissue in the human body in order to treat a disease, endothelial barrier permeability of the drug should be secured as well as the development of the drug itself. As an example, in the case of the brain microvascular endothelial cell layer, there is almost no caveolae, and the binding between endothelial cells is strong (tight junction), such that permeation of drugs having a large molecular weight such as a protein is substantially impossible.
Therefore, researches into permeability of a material in the endothelial cell layer according to expression or progression of diseases, permeability of drugs in the endothelial cell layer, drugs capable of adjusting permeability of drugs in the endothelial cell layer, drugs capable of recovering an abnormal endothelial cell layer, and the like, should be conducted in medical and pharmaceutical fields. However, substantially, a method capable of measuring permeability of the endothelial cell layer in vitro and measuring and evaluating a change against external stimulation including drugs has been barely suggested.
Meanwhile, dielectric spectroscopy, which is to apply an alternating current (AC) electric field to cells to thereby measure specific dielectric responses according to interfacial polarization patterns, is a method applied in order to observe various cellular functions such as viability or death of a single cell, anabolism and differentiation of cells by endomitosis, cell stress, and the like.
The dielectric spectroscopy may observe living cells in vitro under an environment similar to that in the human body based on electric response to AC stimulation and analyze the status of the living cells. In addition, there is no need for pretreatment of a sample according to a complicated protocol, an expensive apparatus, and drug in the dielectric spectroscopy, etc., such that the dielectric spectroscopy is a significantly useful method in a commercial view.
The present applicant found that permeability of the endothelial cell layer may be precisely measured in vitro by using the dielectric spectroscopy, and a change in the permeability of the endothelial cell layer against external stimulation including drugs may be measured and evaluated, thereby completing the present invention.